True rifampicin resistance missed by the MGIT: prevalence of this pheno/genotype in the UK and Ireland after 18 month surveillance.

OBJECTIVES: To characterize rifampicin-resistant strains missed by the Mycobacteria Growth Indicator Tube (MGIT) 960 system but not by egg-based media in the UK and Ireland and to ascertain their prevalence. METHODS: All strains sent for second-line susceptibility testing were prospectively collected. Drug Susceptibility Testing was performed by Resistance Ratio (RR), Proportion Method (PM), MGIT 960 and MIC determination by microdilution. Rifampicin-resistance-conferring mutations were detected with line probe assays and sequencing. At the end of the study period, retrospective archived strains from 2010 to 2014 showing key mutations were analysed phenotypically and genotypically. RESULTS: Seventeen of 7234 prospective isolates were included. All of them were susceptible by MGIT. One was borderline by RR (MIC to rifampicin of 4 mg/L) and was resistant by PM. Eight were resistant and eight were highly resistant on RR. These 16 isolates had MICs between 1 and 8 mg/L on microdilution. With PM, 16/17 were susceptible to rifampicin. 17/17 had mutations in the rpoB gene. D516Y was the mutation most frequently found (13/17). Retrospectively, ten additional strains with key genotypes were found in our collection: 6/10 were susceptible in the MGIT and resistant in RR. Of the 27 studied strains, the MGIT only detected resistance in four. CONCLUSIONS: Rifampicin resistance is missed by the MGIT system. In the UK and Ireland the prevalence of these strains is low. The introduction of routine molecular testing would detect false susceptibility. Further research is needed to ascertain the role of these strains in clinical failure and their prevalence in other settings.

Mycobacterium chelonae infection: a complication of tattooing.

We describe an outbreak of Mycobacterium chelonae infection in four young immunocompetent patients who were tattooed by the same artist. All had been previously tattooed without complication, but following the latest tattooing session, they all developed a very similar papular eruption confined to skin that had been newly coloured light grey. On histological examination of the eruption, granulomatous inflammation with microabscess formation was seen, in association with the tattoo pigment. Skin cultures grown under optimal conditions grew M. chelonae, sensitive to clarithromycin, from one patient. M. chelonae was also cultured from the contents and nozzle of an opened bottle of light-grey ink from the tattoo parlour frequented by the patients. Dermatologists should consider mycobacterial infection in patients who develop inflammatory changes within a new tattoo.

Rapid organism identification from Bactec NR blood culture media in a diagnostic microbiology laboratory.

AIMS: To evaluate rapid organism identification on positive blood culture Bactec NR media (phial types 26, 27, 42 and 17), and to assess the usefulness of these procedures in a diagnostic microbiology laboratory. METHODS: Two hundred and sixty, first positive, blood culture bottles from individual patients were tested by rapid identification methods selected on the basis of Gram film organism morphology. Tube coagulase and latex agglutination were applied to presumptive staphylococci; latex agglutination antigen detection methods to suspected pneumococci, Neisseria and Haemophilus sp; and latex agglutination grouping tests for cultures thought to be non-pneumococcal streptococci. RESULTS: Media type did not influence test performance (p > 0.05 for all comparisons). Misapplication of methods occurred on eight occasions and there were 14 false positive results, nine involving the latex reagents for group C streptococci and pneumococci. The positive predictive values for tube coagulase tests and latex reactions for H influenzae type b, and N meningitidis groups B and C were 100%. The pneumococcal and staphylococcal latex tests gave positive predictive values of 94.1% and 62.5%, respectively, and the corresponding figure for streptococcal grouping reactions was 75.9%. With the exception of staphylococcal latex testing (80%) all investigation negative predictive values were > 90%. CONCLUSIONS: The performance of the staphylococcal latex agglutination method was unsatisfactory and it is not appropriate for use with the media studied. In view of the cross-reactions observed with the tests used to identify group C streptococci and pneumococci, positive findings must be interpreted with caution. In all other regards the protocol evaluated produced rapid, reliable, clinically useful information and, subject to local experience, is recommended to users of Bactec NR media.

Classification of Bacteroides nodosus by agglutination tests.

One thousand two hundred and sixty seven isolates of Bacteroides nodosus from 292 sheep in 58 flocks were examined. Of these, 1260 could be classified by slide agglutination into 8 serogroups designated A to H. Up to 6 serogroups were detected in individual flocks, with up to 4 serogroups being detected in a single foot. Of the 292 sheep examined, 38 (13%) carried mixed serogroup infections. Determination of the range of serological types infecting a flock frequently required the examination of a number of isolates from each of a number of sheep. Cross-tube agglutination tests carried out on 44 isolates and their antiserums indicated that members of some serogroups could be divisible into subgroups or serotypes. These results suggested that 16 or more serotypes of B. nodosus might exist. The nature of the antigens responsible for both slide and tube agglutination reactions needs to be determined.

Evaluation of surface components of Brucella ovis as antigens for the detection of precipitin antibody in serums from artificially exposed rams.

Surface components of Brucella ovis obtained by gentle physical shearing were tested as a potentially useful source of reagent for selective serological diagnosis. These antigens were used in a radial immunodiffusion (RID) test against serum from rams which had been inoculated with infective semen containing B. ovis by one of 4 routes namely mating rams with ewes previously inoculated intravaginally with infective semen, or by direct inoculation in the prepuce, rectum or nasal passage. Loosely attached surface antigens in the RID test formed precipitin bands with serums collected from rams 2 and 10 weeks after inoculation. In contrast, a detergent extracted membrane antigen B developed precipitin bands only with serum collected 10 weeks after inoculation from rams confirmed bacteriologically to be infected with B. ovis in the genital tract. The route by which the rams were artificially exposed did not affect the outcome of the RID test using the membrane B antigen. However, all experimentally exposed rams had demonstrable CF titres when a heat extracted antigen was used.

Inhibition of calmodulin-activated cyclic nucleotide phosphodiesterase: multiple binding-sites for tricyclic drugs on calmodulin.

A cyclic nucleotide phosphodiesterase from guinea-pig heart is activated by calmodulin in the presence of calcium ions. Activation was measured over a range of calmodulin concentrations, and is antagonised by several tricyclic psychotropic drugs including trifluoperazine, imipramine, chlorpromazine and amitriptyline. When the concentration of amitriptyline was increased, its apparent inhibition constant for binding to calmodulin decreased. This was due in part to binding of amitriptyline to glass surfaces; but after correction for this the discrepancy was still significant. It is proposed that this is due to two sites on calmodulin for amitriptyline, with binding to either site being sufficient to prevent calmodulin from activating phosphodiesterase.

Laboratory diagnosis of bovine tuberculosis.

Lesions of suspected bovine tuberculosis were examined by culture, histopathology and auramine-O (AO) stained smears and the findings correlated with field aspects of the disease. Of 642 lesions considered to be tuberculous, 62.0% yielded M. bovis and 4.5% other mycobacteria (OM). M. bovis and OM were recovered also from 0.6% and 3.6% respectively of 165 cattle which gave tuberculin reactions but had no visible lesions at slaughter. Of 262 lesions in which a histopathological diagnosis other than tuberculosis was made, 1.5% and 3.0% yielded M. bovis and OM respectively. All OM isolates tested belonged to the Mycobacterium-avium-intracellulare-scrofulaceum (MAIS) complex with a predominance of serotype 2. A good relationship was found between the recovery of mycobacteria and histopathology but examination of smears revealed 22.0% apparent false negatives. Apparent false negative culture results were also reported for 35.8% of lesions positive on histopathology and smear examination. The majority of herds yielding M. bovis contained reactors to the tuberculin test and many of these had lesions of tuberculosis. In contrast, herds yielding OM seldom contained reactors to the tuberculin test and rarely reactors with tuberculous lesions. The thoracic cavity was the main site of lesions from infections by M. bovis and OM.

Serological classification of Australian strains of Erysipelothrix rhusiopathiae isolated from pigs, sheep, turkeys and man.

154 strains of Erysipelothrix rhusiopathiae from pigs, sheep, turkeys and man were serotyped by using the double diffusion gel precipitation test. Ten of the 18 serotypes were detected in 151 of the strains. Three strains failed to react with any of the type specific antisera. It was found that serotype 1a shared an antigen(s) with serotype 1b, and that serotype 6 shared an antigen(s) with serotype 14. Serotype 2a and 2b were difficult to distinguish. Since serotypes 1 and 2 were isolated from cases of septicaemia in pigs, and since serotypes 1, 2, 4 and 7 were isolated from cases of arthritis, it was suggested that factors other than serotype were important in causing the various forms of swine erysipelas. The fact that the distribution of serotypes 1a, 1b and 2b between septicaemic and arthritic pigs was similar supported the conclusion that arthritis was consequent to bacteraemia. Serotypes 1a, 1b, 2b, 5, 12 and 15 were isolated from cases of arthritis in sheep, and serotypes 1a and 5 from cases of erysipelas in turkeys. Serotype 2b was isolated from a human specimen.

Brucella abortus infection in the bull.

Observations on 2 bulls from a brucella-infected property are reported. Bull 1 gave serological reactions to Br. abortus in both the SAT and CFT from day 0 to day 141. Br. abortus was not recovered from semen and the bull remained clinically normal. The serological status of bull 2 changed from negative to positive to negative over a 203 day period and remained negative for a further 74 days. Semen agglutinins were only detected on one occasion (20 iu). The first clinical sign observed was epididymitis followed by orchitis, which became apparent on day 122. Br. abortus was not recovered from semen but at autopsy, on day 363, Br. abortus biotype 1 was isolated from the right seminal vesicle and ampulla. The histology of the lesion is described. The literature relating to Br. abortus infection in the bull is discussed. Recommendations are made regarding the diagnosis of Br. abortus infection in bulls.